Impacts of iron on phosphate starvation-induced root hair growth in Arabidopsis

In Arabidopsis, phosphate starvation (-Pi)-induced responses of primary root and lateral root growth are documented to be correlated with ambient iron (Fe) status. However, whether and how Fe participates in -Pi-induced root hair growth (RHG) remains unclear. Here, responses of RHG to different Fe concentrations under Pi sufficiency/deficiency were verified. Generally, distinct dosage effects of Fe on RHG appeared at both Pi levels, due to the generation of reactive oxygen species. Following analyses using auxin mutants and the phr1 mutant revealed that auxin and the central regulator PHR1 are required for Fe-triggered RHG under −Pi. A further proteomic study indicated that processes of vesicle trafficking and auxin synthesis and transport were affected by Fe under −Pi, which were subsequently validated by using a vesicle trafficking inhibitor, brefeldin A, and an auxin reporter, R2D2. Moreover, vesicle trafficking-mediated recycling of PIN2, an auxin efflux transporter, was notably affected by Fe under -Pi. Correspondingly, root hairs of pin2 mutant displayed attenuated responses to Fe under -Pi. Together, we propose that Fe affects auxin signalling probably by modulating vesicle trafficking, chiefly the PIN2 recycling, which might work jointly with PHR1 on modulating -Pi-induced RHG.     

Phosphate or phosphite addition promotes the proteolytic turnover of phosphate-starvation inducible tomato purple acid phosphatase isozymes

Within 48 h of the addition of 2.5 mM phosphate (HPO42-, Pi) or phosphite (H2PO3-, Phi) to 8-day-old Pi-starved (-Pi) tomato suspension cells: (i) secreted and intracellular purple acid phosphatase (PAP) activities decreased by about 12- and 6-fold, respectively and (ii) immunoreactive PAP polypeptides either disappeared (secreted PAPs) or were substantially reduced (intracellular PAP). The degradation of both secreted PAP isozymes was correlated with the de novo synthesis of two extracellular serine proteases having M(r)s of 137 and 121 kDa. In vitro proteolysis of purified secreted tomato PAP isozymes occurred following their 24 h incubation with culture filtrate from Pi-resupplied cells. The results indicate that Pi or Phi addition to -Pi tomato cells induces serine proteases that degrade Pi-starvation inducible extracellular proteins.     

Proteomic analysis of the secretome of rice calli

The cell wall and extracellular matrix in higher plants include secreted proteins that play critical roles in a wide range of cellular processes, such as structural integrity and biogenesis. Compared with the intensive cell wall proteomic studies in Arabidopsis , the list of cell wall proteins identified in monocot species is lacking. Therefore, we conducted a large-scale proteomic analysis of secreted proteins from rice. Highly purified secreted rice proteins were obtained from the medium of a suspension of callus culture and were analyzed with multidimensional protein identification technology (MudPIT). As a result, we could detect a total of 555 rice proteins by MudPIT analysis. Based on bioinformatic analyses, 27.7% (154 proteins) of the identified proteins are considered to be secreted proteins because they possess a signal peptide for the secretory pathway. Among the 154 identified proteins, 27% were functionally categorized as stress response proteins, followed by metabolic proteins (26%) and factors involved in protein modification (24%). Comparative analysis of cell wall proteins from Arabidopsis and rice revealed that one third of the secreted rice proteins overlapped with those of Arabidopsis . Furthermore, 25 novel rice-specific secreted proteins were found. This work presents the large scale of the rice secretory proteome from culture medium, which contributes to a deeper understanding of the rice secretome.     

A high-quality secretome of A549 cells aided the discovery of C4b-binding protein as a novel serum biomarker for non-small cell lung cancer

Cancer secretomes are a promising source for biomarker discovery. The analysis of cancer secretomes still faces some difficulties mainly related to the intracellular contamination, which hinders the qualification and follow-up validations. This study aimed to establish a high-quality secretome of A549 cells by using the cellular proteome as a reference and to test the merits of this refined secretome for biomarker discovery for non-small cell lung cancer (NSCLC). Using one-dimensional gel electrophoresis followed by liquid-chromatography tandem mass spectrometry, we comprehensively investigated the secretome and the concurrent cellular proteome of A549 cells. A high-quality secretome consisting of 382 proteins was refined from 889 initial secretory proteins. More than 85.3% of proteins were annotated as secreted and 76.8% as extracellular or membrane-bound. The discriminative power of the lung-cancer associated secretome was confirmed by gene expression and serum proteomic data. The elevated level of C4b-binding Protein (C4BP) in NSCLC blood was verified by enzyme-linked immunosorbent assays (ELISA, p = 6.07e-6). Moreover, the serum C4BP level in 89 patients showed a strong association with the clinical staging of NSCLC. Our reference-experiment-driven strategy is simple and widely applicable, and may facilitate the identification of novel promising biomarkers of lung cancer.     

Proteomic analysis of the small extracellular vesicles and soluble secretory proteins from cachexia inducing and non-inducing cancer cells

Cancer cachexia is a wasting syndrome characterised by the loss of fat and/or muscle mass in advanced cancer patients. It has been well-established that cancer cells themselves can induce cachexia via the release of several pro-cachectic and pro-inflammatory factors. However, it is unclear how this process is regulated and the key cachexins that are involved. In this study, we validated C26 and EL4 as cachexic and non-cachexic cell models, respectively. Treatment of adipocytes and myotubes with C26 conditioned medium induced lipolysis and atrophy, respectively. We profiled soluble secreted proteins (secretome) as well as small extracellular vesicles (sEVs) released from cachexia-inducing (C26) and non-inducing (EL4) cancer cells by label-free quantitative proteomics. A total of 1268 and 1022 proteins were identified in the secretome of C26 and EL4, respectively. Furthermore, proteomic analysis of sEVs derived from C26 and EL4 cancer cells revealed a distinct difference in the protein cargo. Functional enrichment analysis using FunRich highlighted the enrichment of proteins that are implicated in biological processes such as muscle atrophy, lipolysis, and inflammation in both the secretome and sEVs derived from C26 cancer cells. Overall, our characterisation of the proteomic profiles of the secretory factors and sEVs from cachexia-inducing and non-inducing cancer cells provides insights into tumour factors that promote weight loss by mediating protein and lipid loss in various organs and tissues. Further investigation of these proteins may assist in highlighting potential therapeutic targets and biomarkers of cancer cachexia.     

The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea

The rice blast resistance (R) genes Pi2 and Piz-t confer broad-spectrum resistance against different sets of Magnaporthe grisea isolates. We first identified the Pi2 gene using a map-based cloning strategy. The Pi2 gene is a member of a gene cluster comprising nine gene members (named Nbs1-Pi2 to Nbs9-Pi2) and encodes a protein with a nucleotide-binding site and leucine-rich repeat (LRR) domain. Fine genetic mapping, molecular characterization of the Pi2 susceptible mutants, and complementation tests indicated that Nbs4-Pi2 is the Pi2 gene. The Piz-t gene, a Pi2 allele in the rice cultivar Toride 1, was isolated based on the Pi2 sequence information. Complementation tests confirmed that the family member Nbs4-Piz-t is Piz-t. Sequence comparison revealed that only eight amino-acid changes, which are confined within three consecutive LRR, differentiate Piz-t from Pi2. Of the eight variants, only one locates within the xxLxLxx motif. A reciprocal exchange of the single amino acid between Pi2 and Piz-t did not convert the resistance specificity to each other but, rather, abolished the function of both resistance proteins. These results indicate that the single amino acid in the xxLxLxx motif may be critical for maintaining the recognition surface of Pi2 and Piz-t to their respective avirulence proteins.     

水稻恢复系M630稻瘟病抗性改良及其代谢组研究

稻瘟病是水稻的主要病害之一,改良水稻稻瘟病抗性对水稻生产、推广具有重要意义。本研究以携带广谱抗稻瘟病基因Pi9材料9311-Pi9为供体,水稻优良恢复系M630为受体,将杂交、回交与分子标记辅助选择和背景筛选相结合,获得改良恢复系M630-Pi9。主要农艺性状和稻米品质分析显示改良后的M630-Pi9及其杂交组合徽两优630-Pi9与M630和徽两优630相比无明显差异。进一步利用安徽省优势混合生理小种进行苗期稻瘟病抗性鉴定,结果表明,改良后的M630-Pi9相对于M630抗性明显增强;稻瘟病抗性自然病圃鉴定结果发现改良后的恢复系及其杂交组合抗性明显增加。为了更好地了解稻瘟病抗性基因对植株发育的代谢机制,对M630-Pi9和M630受稻瘟病病原菌侵染后的植株进行代谢组分析,结果显示与M630相比,M630-Pi9中有212种代谢物合成受到调控,其中155种含量降低,57种含量增加;与细胞壁有关的物质含量明显增加,对生物体具有毒害和免疫作用的生物碱类代谢物含量显著降低,与生物胁迫相关的黄酮类物质受到不同程度的调控。这些数据表明稻瘟病抗性基因Pi9可能通过调控植物体中代谢物质的变化进而抵御病原体的侵害。创制的新恢复系稻瘟病抗性显著提高,对稻瘟病抗性机制研究提供依据,为水稻抗病、高产育种提供新的种质。     

Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures: implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation, and the integration of glycolysis with nitrogen assimilation.

Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of Pi starvation of Brassica napus suspension cells. Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104-kDa PEPC subunit. To further assess the influence of Pi deprivation on PEPC, the enzyme was purified from Pi-sufficient (+Pi) and Pi-starved (-Pi) cells to electrophoretic homogeneity and final specific activities of 37-40 micromol phosphoenolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +Pi and -Pi PEPCs are both homotetramers composed of an identical 104-kDa subunit. Respective pH-activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to L-malate inhibition were also indistinguishable. Kinetic studies and phosphatase treatments revealed that PEPC of the +Pi and -Pi cells exists mainly in its dephosphorylated (L-malate sensitive) form. Thus, up-regulation of PEPC activity in -Pi cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +Pi cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7.3, marked activation by glucose 6-phosphate and inhibition by L-malate, L-aspartate, L-glutamate, DL-isocitrate, rutin and quercetin was observed. The following paper provides a model for the coordinate regulation of B. napus PEPC and cytosolic pyruvate kinase by allosteric effectors. L-Aspartate and L-glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B. napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.     

Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem

The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homology with any characterized solute transporter, including the family of H(+)-Pi cotransporters identified in plants and fungi. PHO1 shows highest homology with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter-beta-glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is beta-glucuronidase staining of endodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 additional genes showing homology with PHO1. Thus, PHO1 defines a novel class of proteins involved in ion transport in plants.     

Phosphite disrupts the acclimation of Saccharomyces cerevisiae to phosphate starvation.

The influence of phosphite (H2PO3-) on the response of Saccharomyces cerevisiae to orthophosphate (HPO4(2-); Pi) starvation was assessed. Phosphate-repressible acid phosphatase (rAPase) derepression and cell development were abolished when phosphate-sufficient (+Pi) yeast were subcultured into phosphate-deficient (-Pi) media containing 0.1 mM phosphite. By contrast, treatment with 0.1 mM phosphite exerted no influence on rAPase activity or growth of +Pi cells. 31P NMR spectroscopy revealed that phosphite is assimilated and concentrated by yeast cultured with 0.1 mM phosphite, and that the levels of sugar phosphates, pyrophosphate, and particularly polyphosphate were significantly reduced in the phosphite-treated -Pi cells. Examination of phosphite's effects on two PHO regulon mutants that constitutively express rAPase indicated that (i) a potential target for phosphite's action in -Pi yeast is Pho84 (plasmalemma high-affinity Pi transporter and component of a putative phosphate sensor-complex), and that (ii) an additional mechanism exists to control rAPase expression that is independent of Pho85 (cyclin-dependent protein kinase). Marked accumulation of polyphosphate in the delta pho85 mutant suggested that Pho85 contributes to the control of polyphosphate metabolism. Results are consistent with the hypothesis that phosphite obstructs the signaling pathway by which S. cerevisiae perceives and responds to phosphate deprivation at the molecular level.     

Secretome analysis using a hollow fiber culture system for cancer biomarker discovery

Secreted proteins, collectively referred to as the secretome, were suggested as valuable biomarkers in disease diagnosis and prognosis. However, some secreted proteins from cell cultures are difficult to detect because of their intrinsically low abundance; they are frequently masked by the released proteins from lysed cells and the substantial amounts of serum proteins used in culture medium. The hollow fiber culture (HFC) system is a commercially available system composed of small fibers sealed in a cartridge shell; cells grow on the outside of the fiber. Recently, because this system can help cells grow at a high density, it has been developed and applied in a novel analytical platform for cell secretome collection in cancer biomarker discovery. This article focuses on the advantages of the HFC system, including the effectiveness of the system for collection of secretomes, and reviews the process of cell secretome collection by the HFC system and proteomic approaches to discover cancer biomarkers. The HFC system not only provides a high-density three-dimensional (3D) cell culture system to mimic tumor growth conditions in vivo but can also accommodate numerous cells in a small volume, allowing secreted proteins to be accumulated and concentrated. In addition, cell lysis rates can be greatly reduced, decreasing the amount of contamination by abundant cytosolic proteins from lysed cells. Therefore, the HFC system is useful for preparing a wide range of proteins from cell secretomes and provides an effective method for collecting higher amounts of secreted proteins from cancer cells. This article is part of a Special Issue entitled: An Updated Secretome.     

Reconstitution of the partially purified renal phosphate (Pi) transporter.

Proteins from rabbit kidney brush border membranes were solubilized with 1% Nonidet P-40 (crude membrane proteins) and fractionated according to their isoelectric points (pI) by chromatofocusing. The eluate was pooled into three fractions according to the pI of the samples (1, greater than 6.8; 2, 6.8-5.4; 3, 5.4-4.0). The crude membrane proteins as well as the three fractions were reconstituted into liposomes and transport of Pi was measured by a rapid filtration technique in the presence of an inwardly directed K+ or Na+ gradient. Arsenate-inhibitable Na+-dependent transport of Pi was reconstituted into an osmotically active intravesicular space from both the crude membrane proteins and Fraction 1. In contrast, Fractions 2 and 3 were inactive. Treatment of the crude membrane proteins and the three fractions with the method for extracting phosphorin (a Pi-binding proteolipid found in brush border membranes) yielded Mn2+-dependent binding of Pi characteristic of phosphorin only in the extracts from crude membrane proteins and Fraction 1, the same fractions in which Na+-dependent transport of Pi was found in the reconstituted system. When reconstituted into liposomes, phosphorin was, however, unable to yield Na+-dependent transport of Pi. Moreover, we cannot eliminate the possibility that Na+-Pi transport can occur in the absence of phosphorin, since complete recovery of Na+-Pi transport was not achieved. However, the present data showing localization of the recovered binding and transport systems for Pi in the same protein fraction lend support to the hypothesis that phosphorin might be a constituent of the renal Pi transport system. Whether the presence of phosphorin is necessary or accessory for Na+-dependent Pi transport in intact brush border membrane vesicles or in liposomes reconstituted with crude or purified membrane proteins requires further investigation.     

Analytical constraints for the analysis of human cell line secretomes by shotgun proteomics

Human cell line secretome represents a valuable source of therapeutic targets and candidate biomarkers. Secreted proteins found in biological fluids or culture media are by essence highly diluted. Secretome investigation with proteomic approaches is hardly compatible with the high content of proteins found in complete cell culture media. Therefore, many studies are currently done with media containing few or no protein. Such conditions may perturb cell metabolism and proliferation. Here, we compared seventeen different compositions of culture media for the human bronchial epithelial BEAS-2B cell line. Cell viability, proliferation rate and initial protein charge were systematically compared. We have shown that an important difficulty for the proteomic analysis is due to the presence of detergents such as Pluronic F-68 which hinders peptide mass spectrometry. The high glucose containing DMEM medium which is free of proteins was shown to preserve a good viability and proliferation of cells. With this conditioning medium, we identified 81 extracellular proteins in the secretome of BEAS-2B cells. Moreover, to illustrate this approach, we exposed BEAS-2B cells to a low toxic dose of CoCl_2, and found 24 extracellular proteins modulated by cobalt. This study highlights the possible contribution of such proteomic approach in the field of toxicology.     

LEAF TIP NECROSIS1 plays a pivotal role in the regulation of multiple phosphate starvation responses in rice

Although phosphate (Pi) starvation signaling is well studied in Arabidopsis (Arabidopsis thaliana), it is still largely unknown in rice (Oryza sativa). In this work, a rice leaf tip necrosis1 (ltn1) mutant was identified and characterized. Map-based cloning identified LTN1 as LOC_Os05g48390, the putative ortholog of Arabidopsis PHO2, which plays important roles in Pi starvation signaling. Analysis of transgenic plants harboring a LTN1 promoter::β-glucuronidase construct revealed that LTN1 was preferentially expressed in vascular tissues. The ltn1 mutant exhibited increased Pi uptake and translocation, which led to Pi overaccumulation in shoots. In association with enhanced Pi uptake and transport, some Pi transporters were up-regulated in the ltn1 mutant in the presence of sufficient Pi. Furthermore, the elongation of primary and adventitious roots was enhanced in the ltn1 mutant under Pi starvation, suggesting that LTN1 is involved in Pi-dependent root architecture alteration. Under Pi-sufficient conditions, typical Pi starvation responses such as stimulation of phosphatase and RNase activities, lipid composition alteration, nitrogen assimilation repression, and increased metal uptake were also activated in ltn1. Moreover, analysis of OsmiR399-overexpressing plants showed that LTN1 was down-regulated by OsmiR399. Our results strongly indicate that LTN1 is a crucial Pi starvation signaling component downstream of miR399 involved in the regulation of multiple Pi starvation responses in rice.     

Arabidopsis cell wall proteome defined using multidimensional protein identification technology

With the completion of the sequencing of the Arabidopsis genome and the recent advances in proteomic technology, the identification of proteins from highly complex mixtures is now possible. Rather than using gel electrophoresis and peptide mass fingerprinting, we have used multidimensional protein identification technology (MudPIT) to analyse the "tightly-bound" proteome for purified cell walls from Arabidopsis cell suspension cultures. Using bioinformatics for the prediction of signal peptides for targeting to the secretory pathway and for the absence of ER retention signal, 89 proteins were selected as potential extracellular proteins. Only 33% of these were identified in previous proteomic analyses of Arabidopsis cell walls. A functional classification revealed that a large proportion of the proteins were enzymes, notably carbohydrate active enzymes, peroxidases and proteases. Comparison of all the published proteomic analyses for the Arabidopsis cell wall identified 268 non-redundant genes encoding wall proteins. Sixty of these (22%) were derived from our analysis of tightly-bound wall proteins.     

Plasma proteome profiling of a mouse model of breast cancer identifies a set of up-regulated proteins in common with human breast cancer cells

We have applied an in-depth quantitative proteomic approach, combining isotopic labeling extensive intact protein separation and mass spectrometry, for high confidence identification of protein changes in plasmas from a mouse model of breast cancer. We hypothesized that a wide spectrum of proteins may be up-regulated in plasma with tumor development and that comparisons with proteins expressed in human breast cancer cell lines may identify a subset of up-regulated proteins in common with proteins expressed in breast cancer cell lines that may represent candidate biomarkers for breast cancer. Plasma from PyMT transgenic tumor-bearing mice and matched controls were obtained at two time points during tumor growth. A total of 133 proteins were found to be increased by 1.5-fold or greater at one or both time points. A comparison of this set of proteins with published findings from proteomic analysis of human breast cancer cell lines yielded 49 proteins with increased levels in mouse plasma that were identified in breast cancer cell lines. Pathway analysis comparing the subset of up-regulated proteins known to be expressed in breast cancer cell lines with other up-regulated proteins indicated a cancer related function for the former and a host-response function for the latter. We conclude that integration of proteomic findings from mouse models of breast cancer and from human breast cancer cell lines may help identify a subset of proteins released by breast cancer cells into the circulation and that occur at increased levels in breast cancer.